Summary
Autophagy (Greek: Self digestion) is an intracellular process involved in removal of damaged or misfolded proteins or organelles. Damaged or misfolded proteins or organelles are first engulfed in a membraneous structure called autophagosome, and then the autophagosome fuse with lysosome to form autophagolysosome, where the contents are digested. Autophagy is a catabolic process induced during nutritional depletion via phosphatidylinositol 3 kinase pathway. Autophagy is induced in several diseases such as various cancers, heart failure, etc. When autophagy is induced, several autophagic genes are upregulated that help the formation of autophagosome. Several autophagosome specific marker proteins have been identified, among them MAP1LC3-II protein, which is cleaved from MAP1LC3-I, is specifically incorporated into the autophagosomal membrane. The formation of MAP1LC3-II can be analyzed by Western immunoblotting or immunofluorescence. Detailed methods of detection of MAP1LC3-II by Western immunoblotting and immunofluorescence are described.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Cuervo A. M. (2004). Autophagy: many paths to the same end. Mol. Cell. Biochem. 263, 55–72.
Klionsky D. J. and Emr S. D. (2000). Autophagy as a regulated pathway of cellular degradation. Science 290, 1717–21.
Huang J., Klionsky D. J. (2007). Autophagy and human disease. Cell Cycle. 6, 1837–49.
Takagi H., Matsui Y., Sadoshima J. (2007). The role of autophagy in mediating cell survival and death during ischemia and reperfusion in the heart. Antioxid. Redox. Signal. 9, 1373–81.
Shintani T., Klionsky D. J. (2004). Autophagy in health and disease: a double-edged sword. Science 306, 990–5.
Ravikumar B., Vacher C., Berger Z., Davies J. E., Luo S., Oroz L. G., Scaravilli F., Easton D. F., Duden R., O’Kane C. J., Rubinsztein D. C. (2004). Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat. Genet. 36, 585–95.
Xue L., Fletcher G. C., Tolkovsky A. M. (2001). Mitochondria are selectively eliminated from eukaryotic cells after blockade of caspases Âduring apoptosis. Curr. Biol. 11, 361–5.
Cuervo A. M., Dice J. F. (1996). A receptor for the selective uptake and degradation of proteins by lysosomes. Science 273, 501–3.
Petiot A., Ogier-Denis E., Blommaart E. F., Meijer A. J., Codogno P. (2000). Distinct classes of phosphatidylinositol 3’′-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells. J. Biol. Chem. 275, 992–8.
Klionsky D. J., Cregg J. M., Dunn W. A., Jr., Emr S. D., Sakai Y., Sandoval I. V., Sibirny A., Subramani S., Thumm M., Veenhuis M., Ohsumi Y. (2003). A unified nomenclature for yeast autophagy-related genes. Dev. Cell 5, 539–45.
Kuma A., Mizushima N., Ishihara N., Ohsumi Y. (2002). Formation of the approximately 350-kDa Apg12-Apg5.Apg16 multimeric complex, mediated by Apg16 oligomerization, is essential for autophagy in yeast. J. Biol. Chem. 277, 18619–25.
Tanida I., Ueno T., Kominami E. (2004). LC3 conjugation system in mammalian autophagy. Int. J. Biochem. Cell Biol. 36, 2503–18.
Kirisako T., Ichimura Y., Okada H., Kabeya Y., Mizushima N., Yoshimori T., Ohsumi M., Takao T., Noda T., Ohsumi Y. (2000). The reversible modification regulates the membrane-binding state of Apg8/Aut7 essential for autophagy and the cytoplasm to vacuole targeting pathway. J. Cell Biol. 151, 263–76.
Mizushima N., Yamamoto A., Hatano M., Kobayashi Y., Kabeya Y., Suzuki K., Tokuhisa T., Ohsumi Y., Yoshimori T. (2001). Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells. J. Cell Biol. 152, 657–68.
Mizushima N. (2004). Methods for monitoring autophagy. Int. J. Biochem. Cell Biol. 36, 2491–502.
Biederbick A., Kern H. F., Elsasser H. P. (1995). Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles. Eur. J. Cell Biol. 66, 3–14.
Dice J. F. (2007). Chaperone-mediated autophagy. Autophagy 3y, 295–9.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Gurusamy, N., Das, D.K. (2009). Detection of Cell Death by Autophagy. In: Erhardt, P., Toth, A. (eds) Apoptosis. Methods in Molecular Biology, vol 559. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-017-5_7
Download citation
DOI: https://doi.org/10.1007/978-1-60327-017-5_7
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60327-016-8
Online ISBN: 978-1-60327-017-5
eBook Packages: Springer Protocols